Lb2Cas12a and its engineered variants mediate genome editing in human cells

FASEB J. 2021 Apr;35(4):e21270. doi: 10.1096/fj.202001013RR.

Abstract

Cas12a-mediated targeted genome engineering strategies have enabled a broad range of research and clinical applications. However, the limited target-selection spectrum and low activity/fidelity remain a bottleneck for its widespread application in precision site-specific human genome editing. Therefore, there exists an acute need to identify novel Cas12a nucleases with improved features for genome editing. By screening a range of candidate Cas12a nucleases, here we demonstrate that Lb2Cas12a possesses genome editing activity in human cells and it has greater flexibility in PAM (5'-BYYV-3') selection. Furthermore, we engineered Lb2Cas12a to generate variants (Lb2Cas12a-RVR and Lb2Cas12a-RR), which greatly expands the target-selection spectrum. Our study illustrated that Lb2Cas12a could be harnessed as additional genome editing tool for the manipulation of human genome.

Keywords: PAM; RS1; activity; fidelity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics*
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Cas Systems*
  • Endodeoxyribonucleases / genetics*
  • Gene Editing*
  • Gene Expression Regulation
  • Genome, Human
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Protein Engineering*

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Cas12a protein
  • Endodeoxyribonucleases